biostudies-literatureMcGee JPThermo Fisher ScientificNIA NIH HHSNIDA NIH HHSNIH Office of the DirectorNational Institute of General Medical SciencesNational Institute on Drug AbuseNational Institute on AgingNIH HHSNIGMS NIH HHS2723-2727https://www.ebi.ac.uk/biostudies/studies/S-EPMC7878367biostudies-literatureUnknown93(5)Native mass spectrometry involves transferring large biomolecular complexes into the gas phase, enabling the characterization of their composition and stoichiometry. However, the overlap in distributions created by residual solvation, ionic adducts, and post-translational modifications creates a high degree of complexity that typically goes unresolved at masses above ∼150 kDa. Therefore, native mass spectrometry would greatly benefit from higher resolution approaches for intact proteins and their complexes. By recording mass spectra of individual ions via charge detection mass spectrometry, we report isotopic resolution for pyruvate kinase (232 kDa) and β-galactosidase (466 kDa), extending the limits of isotopic resolution for high mass and high <i>m</i>/<i>z</i> by >2.5-fold and >1.6-fold, respectively.Analytical chemistryIsotopic Resolution of Protein Complexes up to 466 kDa Using Individual Ion Mass Spectrometry.PMC7878367RF1 AG063903S10 OD025194F31 AG069456P41 GM108569P30 DA018310Senko MWMelani RDKafader JOMcGee JPYip PFKelleher NLCompton PDfalseIsotopic Resolution of Protein Complexes up to 466 kDa Using Individual Ion Mass Spectrometry.Native mass spectrometry involves transferring large biomolecular complexes into the gas phase, enabling the characterization of their composition and stoichiometry. However, the overlap in distributions created by residual solvation, ionic adducts, and post-translational modifications creates a high degree of complexity that typically goes unresolved at masses above ∼150 kDa. Therefore, native mass spectrometry would greatly benefit from higher resolution approaches for intact proteins and their complexes. By recording mass spectra of individual ions via charge detection mass spectrometry, we report isotopic resolution for pyruvate kinase (232 kDa) and β-galactosidase (466 kDa), extending the limits of isotopic resolution for high mass and high <i>m</i>/<i>z</i> by >2.5-fold and >1.6-fold, respectively.2021-01-01T00:00:00Z2021 Feb2025-04-04T00:38:59.392Z2025-04-04T00:38:59.392ZS-EPMC78783673332289310.1021/acs.analchem.0c03282